Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthetic pathway for catecholamine synthesis. Stress triggers an increase in TH activity, resulting in increased release of catecholamines from both neurons and the adrenal medulla. In response to stress three phases of TH activation have been identified (acute, sustained and chronic) and each phase has a unique mechanism. The acute and chronic phases have been studied in vivo in a number of animal models, but to date the sustained phase has only been characterised in vitro. We aimed to investigate the effects of dual exposure to lipopolysaccharide (LPS) in neonatal rats on TH protein, TH phosphorylation at serine residues 19, 31 and 40 and TH activity in the adrenal gland over the sustained phase. Wistar rats were administered LPS (0.05 mg/kg, intraperitoneal injection) or an equivolume of non-pyrogenic saline on days 3 and 5 postpartum. Adrenal glands were collected at 4, 24 and 48 h after the drug exposure on day 5. Neonatal LPS treatment resulted in increases in TH phosphorylation of Ser40 at 4 and 24 h, TH phosphorylation of Ser31 at 24 h, TH activity at 4 and 24 h and TH protein at 48 h. We therefore have provided evidence for the first time that TH phosphorylation at Ser31 and Ser40 occurs for up to 24 h in vivo and leads to TH activation independent of TH protein synthesis, suggesting that the sustained phase of TH activation occurs in vivo.